Primate immunodeficiency viruses target helper T-cells and macrophages/monocytes through binding of the viral envelope glycoprotein to a combination of CD4 and a chemokine receptor (CCR4 or CXCR5) on the surface of the host cells. Strikingly, infection results in rapid and sustained downregulation of CD4 and, to a lesser extent, the chemokine receptors. Downregulation of these viral co-receptors prevents superinfection, promotes virion release and interferes with the immune response, leading to the establishment of a robust infection. CD4 downregulation is so important to the life cycle of human immunodeficiency virus-1 (HIV-1) that two accessory proteins, Nef and Vpu, encoded in the viral genome are devoted to this task. Indeed, Nef and Vpu are critical for the progression from infection to AIDS, a fact that is best illustrated by the existence of long-term non-progressors that are infected with HIV-1 strains bearing inactivating mutations in the genes encoding these proteins. Therefore, pharmacologic or biologic perturbation of Nef and/or Vpu has the potential to prevent the pathogenic effects of HIV-1. To date, however, this potential has not been realized mainly because Nef and Vpu have no enzymatic activity and their mechanisms of action are insufficiently understood. In previous work, we made substantial progress towards elucidating the mechanism of CD4 downregulation by Nef. We found that Nef connects surface CD4 to both the endocytic and lysosomal targeting machineries, leading to efficient and sustained removal of CD4 from the host cells early during infection. The current project focuses on the mechanisms by which Vpu downregulates CD4 at later stages of infection. Vpu is a small transmembrane protein comprising a short luminal domain, a single transmembrane domain (TMD) and a cytosolic domain. The Vpu cytosolic domain simultaneously binds to the CD4 cytosolic tail and the SCF-beta-TrCP E3 ubiquitin ligase complex, causing CD4 ubiquitination and its subsequent targeting for degradation by the proteasome. Our studies revealed the following novel aspects of this process: (i) degradation involves at least some components of the cellular ER-associated degradation (ERAD) machinery, including the VCP-UFD1L-NPL4 dislocase complex;(ii) CD4 ubiquitination depends on not only lysine but also serine and threonine residues in the CD4 tail;(iii) Vpu mediates CD4 retention in the ER in addition to targeting to ERAD, and (iv) the transmembrane domain of Vpu is required for both ER retention and ERAD targeting of CD4. The multiple levels at which Vpu engages the cellular quality control mechanisms underscore the importance of ensuring profound suppression of CD4 to the life cycle of HIV-1. A surprising finding of our studies on Vpu-induced CD4 downregulation was the requirement of serine and threonine residues for CD4 ubiquitination and targeting to ERAD. To determine how common this requirement is, we examined the degradation of a prototypical ERAD substrate, the alpha subunit of the T-cell antigen receptor complex (TCR-alpha). TCR-alpha is a type I integral membrane protein that becomes ubiquitinated and targeted to ERAD when it fails to assemble into the complete TCR complex. Remarkably, TCR-alpha has a cytosolic tail of only five amino acid residues (i.e., RLWSS), none of which is the canonical ubiquitin-acceptor lysine. We found that substitution of two conserved serine residues in the cytosolic tail of TCR-alpha to alanine decreased ubiquitination, whereas placement of additional serine residues enhanced it. Moreover, replacement of the cytosolic serine residues by other ubiquitinatable residues (i.e., cysteine, threonine, or lysine) allowed ubiquitination to take place. Serine-dependent ubiquitination perfectly correlated with targeting of TCR-alpha for ERAD. We also found that this ubiquitination is mediated by the ER-localized ubiquitin ligase, HRD1. These findings indicated that serine-dependent, HRD1-mediated ubiquitination targets TCR-alpha to the ERAD pathway. Thus, Vpu does not induce a viral-specific modification but exploits an endogenous machinery for serine-dependent ubiquitination in order to downregulate CD4.